Involvement of the TNF and FasL Produced by CD11b Kupffer Cells/Macrophages in CCl4-Induced Acute Hepatic Injury

We previously reported that F4/80+ Kupffer cells are subclassified into CD68+ Kupffer cells with phagocytic and ROS producing capacity, and CD11b+ Kupffer cells with cytokine-producing capacity. Carbon tetrachloride (CCl4)-induced hepatic injury is a well-known chemical-induced hepatocyte injury. In the present study, we investigated the immunological role of Kupffer cells/macrophages in CCl4-induced hepatitis in mice. The immunohistochemical analysis of the liver and the flow cytometry of the liver mononuclear cells showed that clodronate liposome (c-lipo) treatment greatly decreased the spindle-shaped F4/80+ or CD68+ cells, while the oval-shaped F4/80+ CD11b+ cells increased. Notably, severe hepatic injury induced by CCl4 was further aggravated by c-lipo-pretreatment. The population of CD11b+ Kupffer cells/macrophages dramatically increased 24 hour (h) after CCl4 administration, especially in c-lipo-pretreated mice. The CD11b+ Kupffer cells expressed intracellular TNF and surface Fas-ligand (FasL). Furthermore, anti-TNF Ab pretreatment (which decreased the FasL expression of CD11b+ Kupffer cells), anti-FasL Ab pretreatment or gld/gld mice attenuated the liver injury induced by CCl4. CD1d−/− mouse and cell depletion experiments showed that NKT cells and NK cells were not involved in the hepatic injury. The adoptive transfer and cytotoxic assay against primary cultured hepatocytes confirmed the role of CD11b+ Kupffer cells in CCl4-induced hepatitis. Interestingly, the serum MCP-1 level rapidly increased and peaked at six h after c-lipo pretreatment, suggesting that the MCP-1 produced by c-lipo-phagocytized CD68+ Kupffer cells may recruit CD11b+ macrophages from the periphery and bone marrow. The CD11b+ Kupffer cells producing TNF and FasL thus play a pivotal role in CCl4-induced acute hepatic injury.


Introduction
Carbon tetrachloride (CCl 4 ) is a highly toxic chemical agent that induces acute hepatic injury, while chronic administration of CCl 4 induces fibrosis, cirrhosis and carcinogenesis. Although chronic CCl 4 injection models have been extensively studied as liver fibrosis and cirrhosis models [1][2][3][4][5], the acute phase of this hepatitis has been less characterized. The acute phase of CCl 4 hepatic injury may be produced by the formation of reactive oxygen species (ROS) in the endoplasmic reticulum of hepatocytes by cytochrome p450 enzymes, which may also induce mitochondrial dysfunction, including changes in calcium homeostasis, energy production and the beta-oxidation of fatty acids, all of which can result in hepatocyte damage [4,6,7]. However, although a role for Kupffer cells has been suggested [2,[8][9][10], the immune mechanism involved in the acute phase of CCl 4 -induced hepatic injury has not been extensively examined.
It is now generally accepted that the livers of mice and humans contain various kinds of innate immune cells [11][12][13]. It is well known that liver NK cells and NKT cells potently produce IFN-c in response to IL-12 and/or LPS [11][12][13]. Interestingly, liver B cells (mostly B-2 cells) produce IL-12 and IFN-c but not IgM, in response to LPS (vice versa for spleen B cells) [14]. Furthermore, these IL-12-producing liver B cells, in contrast to spleen B cells, phagocytose bacteria and kill them [15,16]. Therefore, these liver immune cells, including B cells and their cytokines, primarily act as innate immune effectors against infections and tumors by their T helper-1 immune response in the liver. However, they also sometimes induce hepatic injury, septic shock and multi-organ failure [12,13,17]. In addition, we have recently reported that liver F4/80 + Kupffer cells/macrophages can be subclassified almost exclusively into two different subsets; a CD68 + subset with phagocytic, ROS production and bactericidal capacities, and a CD11b + subset with cytokine (TNF and IL-12) production and antitumor capacities [12,13,18,19].
Gadolinium chloride (GdCl 3 ) and clodronate liposomes (c-lipo), are both cytotoxic to Kupffer cells, and have been used to deplete Kupffer cells in rodents. Some reports have suggested that GdCl 3 and c-lipo completely eliminate Kupffer cells based on immunohistochemical examinations. However, we reported and demonstrated herein that these agents deplete only CD68 + Kuppfer cells (resident or fixed), but not CD11b + Kupffer cells, based on the flow cytometric analysis of liver mononuclear cells [18,19]. Consistent with our data, Holt et al. also demonstrated that clipo administration did not eliminate the CD11b high F4/80 low subset, whereas the other CD11b low F4/80 high subset was almost completely depleted [27]. We consider that the former population corresponds to CD11b + Kupffer cells and the latter population corresponds to CD68 + cells in our studies.
In the present study, we demonstrate by immunohistochemistry, as well as flow cytometry, that the large and spindle-shaped CD68 + cells were indeed depleted by c-lipo treatment, whereas the small and round-shaped CD11b + population increased. Furthermore, CD11b + Kupffer cells play an important role in the acute phase of CCl 4 -induced hepatitis as a result of their production of TNF and FasL, which occurs in an NKT cell-independent manner. In addition, the c-lipo-phagocytized CD68 + Kupffer cells were found to produce monocyte chemoattractant protein (MCP)-1, and lead to the subsequent accumulation of CD11b + Kupffer cells/macrophages into the liver before CCl 4 injection, which aggravates the hepatic injury induced by CCl 4 injection.

Mice and Reagents
The Ethics Committee of Animal Care and Experimentation, National Defense Medical College, Japan, approved all requests for animals and the intended procedures of the present study (Permission number: 12039).

Induction of Acute Liver Injury
To induce acute liver injury by CCl 4 , mice were injected intraperitoneally with a single dose of CCl 4 (0.6 mg/kg in oil). Control groups received the same volume of vehicle (oil) intraperitoneally.

Isolation of MNCs, Including Kupffer Cells
The murine livers were removed under deep anesthesia. The liver MNCs were prepared essentially as described previously [18]. Briefly, the livers were minced and suspended in HBSS containing 0.05% collagenase (Wako, Osaka, Japan), and then were shaken for 20 min in a 37uC water bath. Next, the liver specimens were washed in 1% FBS RPMI 1640 and then filtered through a stainless steel mesh. After mixing in isotonic 33% Percoll solution containing heparin, the samples were centrifuged for 15 min at 5006g at room temperature. After removing the supernatant, the pellets were resuspended in a red blood cell lysis solution and then were washed twice in 10% FBS RPMI 1640.

Pathological Examination
For pathological examinations, the mice were euthanized prior to removal of their livers. The liver was then immersed in 10% formalin for two days. Slides were prepared from these specimens and stained with hematoxylin and eosin.

Intracellular Staining for TNF
MNCs were incubated with BD GolgiStop (0.7 mg/ml, BD PharMingen) for three h before staining. After incubation with Fcblocker, the cells were stained with a FITC-conjugated anti-F4/80 Ab and Cy5-conjugated anti-CD11b Ab or a biotin-conjugated anti-CD68 Ab with Cy5-streptavidin. Subsequently, the cells were incubated with BD Cytofix/Cytoperm solution (BD Pharmingen) at 4uC for 20 min and then washed with BD Perm/Wash solution (BD Pharmingen). Thereafter, the cells were stained with a PEconjugated anti-TNF mAb (eBioscience) or isotype rat IgG1 Ab (eBioscience) at 4uC for 20 min and then analyzed using the FC500 instrument.

Measurement of the Alanine Amino Transferase, Cytokine and MCP-1 Levels
The serum alanine amino transferase (ALT) level was measured using a DRICHEM 3000V instrument (Fuji Medical Systems, Tokyo). ELISA kits for TNF (BD Biosciences, San Jose, CA, USA) and MCP-1 (R&D system, San Jose, CA, USA) were used to analyze the levels of these cytokines.
Isolation of F4/80 + CD11b + Kupffer Cells from CCl 4 Treated Mice using MACS Sort System Livers were obtained from mice 14 h after the injection of CCl 4 , and minced liver specimens without collagenase treatment in 1% FBS RPMI 1640 were filtered through a stainless steel mesh. Thereafter, the liver MNCs were obtained using a 33% Percoll solution. The MNCs were stained with PE-Cy5 labeled anti-F4/80 antibody following conjugation with anti-PE magnetic beads (Miltenyi Biotec GmbH). Beads conjugated F4/80 + cells (which were also positive for PE-Cy5) or F4/80 2 cells were magnetically sorted by Super MACS system (Miltenyi Biotec GmbH). Adoptive Transfer Experiments MACS sorted 5610 6 F4/80 + cells (PE-Cy5 + ) or 5610 6 F4/80 2 cells were adoptively transferred into normal mice or CD68 + Kupffer cell-depleted (by c-lipo) mice. As an experimental control, liver 5610 6 F4/80 + cells from oil-treated mice were transferred into normal mice. After adoptive transfer, recruitment of transferred F4/80 + cells into recipient liver was confirmed by the presence of PE-Cy5 positive cells. The induction of liver injury after transfer was analysed and compared within each group.

In vitro Cytotoxic Assay Against Primary Cultured Hepatocytes
Primary cultured hepatocytes were used as target cells. Hepatocytes were obtained from 8 week of age B6 mice essentially described previously [21]. In brief, liver were perfused with collagenase from portal vein, and dispersed hepatocytes were suspended in hepatocyte growth medium (HCGM) and seeded into collagen type I coated 96 well plate (Iwaki, Funabashi, Japan) with 2.0610 3 cells/well concentration. After 12 h of incubation, hepatocytes adhered to the bottom of the plate and medium was changed by HCGM containing 10 mCi of Na 2 51 Cr0 4 /ml and incubated additional 12 h. The 51 Cr labeled hepatocytes were washed three times with HCGM, and effector cells were added following 4 h of incubation. F4/80 + cells (which were also positive for PE-Cy5) or F4/80 2 cells were obtained by MACS system as described above. The concentration of effector MNCs were adjusted to 5.0610 5 cells/well (250:1) and 2.5610 5 cells/well 125:1). Culture supernatants were harvested and radio activities were measured by gamma counter.

Statistical Analysis
The results are expressed as the mean values 6 SE. The statistical analyses were performed using a GraphPad Prism 5 software package (GraphPad Software, La Jolla, CA). Statistical evaluations were compared using the standard one-way analysis of variance followed by the Bonferroni post-hoc test. A value of P, 0.05 was considered to be significant.

Depletion of CD68 + Kupffer Cells and Aggravation of CCl 4-Induced Hepatic Injury by c-lipo Pretreatment
We previously reported that c-lipo or GdCl 3 selectively depleted only CD68 + Kupffer cells, but increased the population of CD11b + Kupffer cells/macrophages, as determined by flow cytometry [18]. Indeed, 36 h after c-lipo treatment, an immunohistochemical analysis showed that the spindle-shaped CD68 + cells and F4/80 + cells were greatly decreased in the liver, while the oval-shaped CD68 + or F4/80 + cells still remained, and the population of CD11b + cells appeared to increase (Fig. 1). A flow cytometric analysis also confirmed that c-lipo treatment proportionally decreased the liver CD68 + Kupffer cells (41% to 12.8%, Fig. 2 right panels) but that the liver CD11b + Kupffer cells/ macrophages increased (37% to 75.7%, Fig. 2 right panels). Of note, the number of spleen, bone marrow and peripheral blood CD11b + monocytes/macrophages did not decrease [19]. A forward scatter (FS) and side scatter (SS) analysis revealed that the CD68 + Kupffer cells are relatively large and show a complex structure, and most of them disappeared following c-lipo treatment (blue dots, Fig. 2, left panels). In contrast, the CD11b + Kupffer cells/macrophages are small and have a simple structure (red dots, Fig. 2, left panels). The CD11b + Kupffer cells/macrophages became larger after c-lipo treatment, as indicated by the FS analysis (Fig. 2, left panels, FS values; 43666.8 vs 40162.9, n = 5, p,0.05), suggesting that they were activated after c-lipo treatment. However, the total number of liver MNCs yielded from the liver did not significantly change following the c-lipo treatment (approximately 7610 6 /liver). Thus, the number of CD68 + Kupffer cells decreased, while the number of CD11b + Kupffer cells/ macrophages increased, in the liver.
CCl 4 injection induced severe hepatic injury, as indicated by the ALT levels, and the hepatic injury was aggravated in c-lipo pretreated mice (Fig. 3A). Consistently, the TNF levels in c-lipo pretreated mice after CCl 4 injection were higher than those of control mice (Fig. 3B). Twenty-four h after CCl 4 injection, the population of F4/80 + CD11b + Kupffer cells was greatly increased compared to that in control oil-injected mice, and c-lipo-pretreatment further increased the number of F4/80 + Kupffer cells after CCl 4 injection (Fig. 4). However, the number of CD68 + Kupffer cells was reduced, especially in c-lipo-pretreated mice, after CCl 4 injection. In addition, the livers of c-lipo-pretreated mice showed more and larger necrotic areas after CCl 4 -injection than did the PBSpretreated control mice (Fig. 5).

The Role of TNF, FasL, NKT Cells and NK Cells in CCl 4induced Hepatic Injury
Since we previously reported that TNF and/or FasL are involved in the pathogenesis of some experimental models of hepatitis [17,21,23], we examined the functions of TNF and FasL in CCl 4 -induced hepatitis. Pretreatment of mice with a neutralizing anti-TNF Ab or anti-FasL Ab significantly decreased the serum ALT levels after CCl 4 administration (Fig. 6A). However, pretreatment with a neutralizing anti-IFN-c Ab did not affect the serum ALT levels after CCl 4 administration (Fig. 6A). Additionally, liver injury was significantly ameliorated in FasL deficient gld mice (Fig. 6B). These results suggest that TNF and FasL, but not IFN-c, are profoundly involved in the CCl 4-induced acute hepatic injury.
Since we previously reported that NKT cells are responsible for the hepatic injury induced by a-GalCer or bacterial DNA motifs [17,21,23], we next examined the effects of CCl 4 on the CD1d2/2 mice. Since CD1d2/2 mice on a B6 background were not commercially available, we used CD1d2/2 mice on a BALB/c background. The results showed that the serum ALT levels were not significantly different between CD1d2/2 mice and control mice (Fig. 6C). Next, we examined the effect of CCl 4 on mice depleted of NK cells by an anti-AGM1 Ab, or depleted of NKT cells as well as NK cells (by an anti-NK1.1 Ab), and found that the serum ALT levels were similar to those in control mice (Fig. 6D). These results suggest that NK/NKT cells are not involved in the CCl 4 -induced acute hepatic injury.

Intracellular TNF Production and FasL Expression of Kupffer Cells, and the Effects of an anti-TNF Ab
We next examined the intracellular TNF production and surface FasL expression of liver MNCs 12 h after CCl 4 injection. The F4/80 + CD11b + Kupffer cells, but not other cells, including F4/80 + CD68 + Kupffer cells, produced TNF and expressed FasL (Figs. 7A, B). The staining of F4/80 + CD11b + Kupffer cells for TNF was lower at six and 24 h after CCl 4 injection compared to that at 12 h after CCl 4 injection (not shown). We further examined the relationship between the TNF and FasL expression of F4/80 + CD11b + Kupffer cells. Pretreatment with a neutralizing TNF Ab dramatically decreased the FasL expression of the F4/80 + CD11b + Kupffer cells (Fig. 7C). These results suggest that the TNF produced by F4/80 + CD11b + Kupffer cells induces their FasL expression, and that F4/80 + CD11b + Kupffer cells play a crucial role in CCl 4 -induced acute hepatic injury via TNF/FasL.

Induction of Hepatic Injury in Mice by the Adoptive Transfer of CD11b + Kupffer Cells from mice Injected with CCl 4
We previously reported that the hepatic MNCs obtained without collagenase digestion contain many CD11b + Kupffer cells but few CD68 + Kupffer cells [18]. Consistently, the MACS-sorted liver F4/80 + cells without collagenase digestion from CCl 4injected mice were primarily F4/80 + CD11b + Kupffer cells (85%, Fig. 8A). A major proportion of the F4/80 -CD11b high cells were Gr1-positive neutrophils (62%, Fig. 8A and not shown) and the CD11b -F4/80 2 cells were lymphocytes (30%, Fig. 8A and not shown). Moreover, MACS sorted F4/80 + CD11b + Kupffer cells were adoptively transferred into normal mice or CD68 + cell depleted mice, and serum ALTs were examined. The hepatic injury induced by transferred F4/80 + CD11b + Kupffer cells was stronger than that induced by transferred F4/80 2 cells (Fig. 8B). Moreover, when F4/80 + CD11b + Kupffer cells from CCl 4 -injected mice were transferred into mice depleted of CD68 + Kupffer cells (36 h after c-lipo injection), a more severe hepatic injury was evoked than that in mice without c-lipo pretreatment (Fig. 8B). These results raise the possibility that resident CD68 + Kupffer cells may normally inhibit the function of CD11b + Kupffer cells/macrophages. However, it should be noted that the transfer of liver F4/80 2 cells also induced a substantial hepatic injury in clodronate-pretreated mice (Fig. 8B), suggesting that the activated neutrophils contained in the F4/80 2 cells may also have hepatotoxicity under in vivo condition. Flow cytometric analysis of recipient liver MNCs at 1.5 h after the adoptive transfer without additional staining showed that 4.5% were positive for PE-Cy5 in mice transferred with F4/80 + cells (Fig. S1, left panel) and few (0.6%) were postive in mice transferred with F4/80 2 cells (nonspecific staining) (Fig. S1, middle panel). FS/SS analysis verified that such positive cells (in left panel) were confirmed to be macrophages (Fig. S1, right panel). Furthermore, MACS sorted F4/80 + CD11b + Kupffer cells from CCl 4 -injected mice showed cytotoxicity against primary cultured hepatocytes in vitro, and this cytotoxicity was effectively blocked by neutralization of FasL, whereas F4/80 2 cells did not showed the killing activity in vitro (Fig. 8C).

C-lipo Treatment of Mice Before CCl 4 Administration Increases the Serum Level of MCP-1
To elucidate the mechanism by which CD11b Kupffer cells are increased by c-lipo pretreatment, the serum MCP-1 levels were monitored after c-lipo injection. MCP-1 is a major chemokine, and is a ligand for CC-chemokine receptor 2 (CCR2). Intriguingly, the serum MCP-1 levels rapidly increased and peaked at six h after c-lipo injection after c-lipo treatment (Fig. 9A). Furthermore, the MCP-1 levels did not increase any more in the c-lipo-pretreated mice after CCl 4 injection (Fig. 9B). These results suggest that the CD68 Kupffer cells are activated after phagocytosing c-lipo, and that they produced MCP-1 and thereafter underwent apoptosis due to the cytotoxicity of clodronate. Our results also suggest that MCP-1 plays a critical role in the recruitment and activation of CD11b Kupffer cells from the periphery or bone marrow.
To confirm the presence of MCP-1-producing CD68 cells, liver MNCs were harvested from mice one hour after CCl 4 administration, and CD68 + cells were purified by the MACS system from liver MNCs obtained from liver specimens with collagenase treatment, and F4/80 + CD11b + cells (F4/80 + cells from mice depleted of CD68 cells by c-lipo) were also obtained by the MACS system. Both subsets were cultured in vitro for the indicated amount of time. The results showed that CD68 cells produced a substantial amount of MCP-1 beyond 12 h after culture, but CD11b cells did not produce any MCP-1 (Fig. 9C). We also found that, in mice depleted of CD68 Kupffer cells injected with viable Staphylococcus aureus, the serum MCP-1 levels (peak at 3 h) were greatly reduced compared to those in control mice, and that CD68 Kupffer celldepleted liver MNCs cultured with bacteria in vitro did not produce any MCP-1 [19]. However, it was considered possible that the MCP-1 produced by CD68 Kupffer cells at 24 h after CCl 4 administration may not be involved in the recruitment of CD11b macrophages/Kupffer cells or hepatic injury.

Discussion
In the current study, we explored a unique immunological mechanism of CCl 4 -induced acute hepatic injury in mice. Namely, CD11b + Kupffer cells produce TNF, as well as FasL, and induce hepatic injury, in which IFN-c, NK cells and NKT cells are not involved. Furthermore, c-lipo pretreatment to deplete CD68 + Kupffer cells promoted MCP-1 production from the CD68 + Kupffer cells, presumably before they underwent apoptosis, which led to the accumulation and activation of CD11b + Kupffer cells, and markedly aggravated the hepatic injury following CCl 4 injection. Both CpG-ODN and a-GalCer-mediated hepatitis are TNF/FasL/Fas pathway-dependent, and the final effectors in We recently demonstrated that CD68 + Kupffer cells are fixed Kupffer cells and cannot be harvested unless collagenase treatment of liver tissues is carried out, whereas CD11b + Kupffer cells are easily obtained without collagenase treatment from liver specimens [18]. In addition, although CD68 + Kupffer cells are mainly located in the mid-zonal region between the portal vein and the central vein, CD11b + Kupffer cells are equally distributed in the liver tissues [18]. Therefore, it was suggested that CD68 + cells are resident Kupffer cells, and that CD11b + cells may be recruited from the periphery or bone marrow to the inflamed liver [18,19]. Furthermore, the functions of these cell subsets are quite different: CD68 + Kupffer cells have phagocytic, ROS-producing and bactericidal activities, while CD11b + Kupffer cells have cytokine (IL-12 and TNF)-producing capacity and are involved in antitumor immunity [18,19]. Our present results also confirmed by immunohistochemistry, as well as flow cytometry, that c-lipo depletes CD68 + Kupffer cells, but increases the number of CD11b + Kupffer cells. Furthermore, resident CD68 + Kupffer cells are radio-resistant, whereas CD11b + Kupffer cells/macrophages are radio-sensitive [19,27] which were reconstituted by the transfer of bone marrow cells [19]. Therefore, they are distinct types of macrophages, and most of the monocytes/macrophages in the spleen and peripheral blood are CD11b + cells, while CD68 + Kupffer cells predominate in the liver. Although intracellular CD68 was present in the cytosol of CD11b + Kupffer cells and may be expressed on the cell surface of the cells upon activation [26], the intra-cellular CD68 expression was still much lower than that   injected with c-lipo or PBS, the sera were obtained at the indicated time points, and the MCP-1 levels were measured (n = 3 in each group). (B) The serum MCP-1 levels did not increase after CCl 4 injection in mice pretreated with c-lipo. The mice pretreated with c-lipo or PBS were injected intraperitoneally with CCl 4 or oil, and sera were obtained at the indicated time points to measure the MCP-1 levels (n = 3 in each group).(C) The ex vivo production of MCP-1 from the liver CD68 + Kupffer cells from CCl 4 -injected mice. One hour after the injection of CCl 4 , the liver MNCs were harvested from the liver by collagenase treatment, and CD68 + cells were obtained by magnetic beads (MACS system). F4/80 + CD11b + cells obtained by F4/80 magnetic beads from the liver MNCs from c-lipo treated mice. Both purified subsets were cultured in vitro for the indicated amounts of time. The data are the means 6 SE from three independent experiments. **p,0.01, *p,0.05 vs. other groups. doi:10.1371/journal.pone.0092515.g009 of CD68 + Kupffer cells, as revealed by flow cytometry (our unpublished observation).
In contrast to the hepatic injury induced by either a-GalCer or CpG-ODN, CD68 + Kupffer cells and their ROS production induced by the TNF produced by CD11b Kupffer cells/ macrophages are the final effectors in Con-A-induced hepatitis [12,13,24]. In all of these types of hepatitis, the TNF was produced by CD11b + Kupffer cells in the early period (at 1 h) after the injection of reagents [17,[20][21][22][23]. However, the serum TNF levels did not start to increase until 12 h after CCl 4 injection, and the intracellular production of TNF in CD11b + Kupffer cells reached a maximum at 12 h after CCl 4 injection, suggesting that TNF may be released from CD11b + Kupffer cells into the systemic circulation beyond 12 h after CCl 4 injection. These results imply that, although CD11b + Kupffer cells are effectors involved in the hepatic injury induced by CCl 4 , CD11b + Kupffer cells may produce TNF and FasL in order to reject the hepatocytes chemically-damaged by CCl 4 to ensure the early termination of hepatic injury. Karlmark et al. also pointed out the marked infiltration of F4/80 + CD11b + Gr1 + macrophages in the acute phase of CCl 4 hepatic injury, and demonstrated the indispensable role of CCR2 (MCP-1 ligand) for their recruitment to the liver [2]. It is plausible that the CD11b + Kupffer cells in our study may be identical to the population reported in their study.
It was previously reported that CCR2-deficient mice showed a dramatic reduction in macrophage accumulation in both the peritoneal cavity and liver upon exposure to inflammatory stimuli [2,35,36]. MCP-1 is a major ligand of CCR2 [36], and is thus an important chemokine that recruits monocytes/macrophages to inflamed organs and tissues. CD68 Kupffer cells may be activated by phagocytosing c-lipo, and may produce MCP-1 before undergoing apoptosis. Although CD68 Kupffer cell-depleted mice did not show an increase in MCP-1 after CCl 4 injection, the hepatic injury, as well as the CD11b + cell recruitment, was enhanced 24 h after CCl 4 injection. These results suggest that, although increased MCP-1 in the early phase (6 h after c-lipopretreatment) may be required to increase and prime CD11b Kupffer cells/macrophages in the liver before CCl 4 administration, other cytokines, including TNF, may be critical for further recruitment of CD11b + cells into the liver and for the resultant hepatic injury after CCl 4 injection.
The time course of the serum MCP-1 levels after the injection of c-lipo or bacteria [19] suggests an important point. When CD11b Kupffer cells are activated and produce TNF in vivo due to exposure to LPS, CpG-ODN and a-GalCer or whole bacteria, the serum TNF levels usually peak at 1 h after administration [17,22,23,37], which is much earlier than the peaks of serum MCP-1 after bacteria or c-lipo injection (3 h or 6 h, respectively). This may be because the production of MCP-1 from CD68 Kupffer cells requires the digestion of c-lipo or bacteria (phagolysosomal formation) [19], whereas CD11b Kupffer cells may rapidly respond to these ligands via TLR-4, 9, or CD1d.
However, further investigations of both CD68 + Kupffer cells and CD11b + Kupffer cells/macrophages and their mutual interactions, as well as their interactions with other liver leukocytes, are required for understanding the chronic inflammation and fibrosis induced by CCl 4 . Figure S1 The recruitment of adoptive transferred F4/ 80 + cells into liver. Liver MNCs were isolated from CCl 4 treated mice and stained with PE-Cy5 labeled anti-F4/80 antibody. F4/80 + and F4/80 2 cells were separated using anti-PE magnetic beads and MACS sorting device. Sorted F4/80 + cells labeled with PE-Cy5, and F4/80 2 cells without labelling, were adoptively transferred into normal mice. Then, liver MNCs obtained from each recipient mouse at 1.5 h after adoptive transfer were analyzed by flow cytometry without additional staining. PE-Cy5 (F4/80) positive cells were counted and depicted in red area and dots. Data are representative of three mice in each group, with similar results. (EPS)